Identification of the mesenchymal stem cell (MSC) population capable of giving rise to odontoblasts secreting dentin during growth (primary dentinogenesis) and repair (reparative dentinogenesis) is critical for the development of improved pulp therapy, regeneration of dentin and ultimately the bioengineering of functional teeth. Despite significant progress in the identification of MSCs within the mouse incisors in vivo, we know much less about MSCs in mouse molars that, unlike incisors, are not continuously growing and are more similar to human dentition. The goal of the present study is to examine the currently controversial roles of the Hedgehog (Hh) signaling pathway and Gli1, a transcription factor that is a downstream effector of the activated canonical Hh signaling pathway in primary and reparative dentinogenesis in mouse molars. Two specific aims have been proposed. Experiments in Aim 1 are designed to test the hypothesis that in growing mice molars, Hh signaling and Gli1 expressing cells in dental mesenchyme give rise to primary odontoblasts. Using lineage tracing and Gli1-CreERT2; Ai9 reporter mice we will examine the contribution of Gli1-Cre-tdTomato expressing cells to primary odontoblasts and the effects of inhibition of Hh signaling by HhAntag, a potent small molecule inhibitor of the Shh pathway, on the activation of Gli1-Cre-tdTomato+ cells and their contribution to primary odontoblasts. Experiments in Aim 2 are designed to test the hypothesis that in adult mice molars, Hh signaling and Gli1 expressing cells in the dental pulp are involved in the formation of second-generation odontoblasts and reparative dentinogenesis. We will perform experimental pulp exposure in adult molars in vivo and examine the expression of Shh ligand and Gli1 after pulp exposure. The contribution of Gli1-Cre-tdTomato expressing cells to second generation of odontoblasts during reparative dentinogenesis and the effects of inhibition of Hh signaling on reparative dentinogenesis and the contribution of Gli1-Cre-tdTomato expressing cells to second generation of odontoblasts will be examined by cell lineage-tracing experiments in GLi1- CreERT2; Ai9 reporter mice. The proposed studies in mouse molars that are not continuously growing and more similar to human dentition will enable us to gain a better understanding of roles of Hh signaling and Gli1 expressing cells in primary and reparative dentinogenesis, an area that has not been studies before.